Journal of Molecular Biology
Volume 272, Issue 3, 26 September 1997, Pages 336-347
Journal home page for Journal of Molecular Biology

Regular article
Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences1

https://doi.org/10.1006/jmbi.1997.1252Get rights and content

Abstract

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

Introduction

Enzymes can be evolved in vitro to exhibit new and useful functions. A sampling of the local sequence space of the enzyme is created by mutagenesis; screening or selection directs the evolution towards the desired features. A successful strategy for improving enzyme activity in non-natural environments (Chen & Arnold, 1993) and on non-natural substrates (Moore & Arnold, 1996) has been to accumulate amino acid substitutions over multiple generations of random mutagenesis and screening. In practice, the best variant identified in each generation is chosen to parent the subsequent generation. Other potentially useful variants are set aside, and their mutations must be rediscovered in the evolved protein background in order to become incorporated. Because there is no mechanism other than back mutation for deleting mutations, this approach can also accumulate deleterious mutations, leading to premature termination of an evolving lineage. These are the classical arguments for the benefits of recombination (sex) in evolution (Maynard Smith, 1988). Recombination allows more rapid accumulation of beneficial mutations present in a population. It also makes possible the removal of deleterious mutations which would otherwise accumulate in an asexual population, a phenomenon known to geneticists as Müller’s ratchet (Müller, 1932). Recombination can provide similar benefits for in vitro molecular evolution Stemmer 1994a, Stemmer 1994b.

Bacillus subtilis p-nitrobenzyl (pNB) esterase catalyzes the hydrolysis of the para-nitrobenzyl esters of various cephalosporin-type antibiotics, a necessary step in their large-scale synthesis (Zock et al., 1994). Using four generations of sequential random mutagenesis and screening, we evolved a series of pNB esterases up to 30 times more active towards hydrolysis of the pNB ester of loracarbef (LCN-pNB) in aqueous dimethylformamide (Moore & Arnold, 1996). During the fourth generation, a large number (∼7500) of pNB esterase clones were screened and partially characterized in order to validate the rapid screening assay. Sixteen improved pNB esterase clones were identified, from which the five most active enzymes (>50% enhancements in activity over the parent enzyme) were characterized. DNA sequencing revealed four unique pNB esterases (Table 1). Due to the limitations of screening, evolved sequences are generated using a low rate of point mutagenesis and typically accumulate a single beneficial mutation per generation. A simple restriction/ligation experiment demonstrated that recombination of mutations present in at least two of those sequences could further improve pNB esterase activity. Recombining gene segments from two improved pNB esterase variants yielded an enzyme twice as active as the best parent. DNA sequencing demonstrated that mutations from each of the two parents were combined in the new sequence (I60V and L334S), while one neutral or slightly deleterious mutation was deleted (K267R; Moore & Arnold, 1996).

Stemmer recently introduced the technique of “DNA shuffling” to create novel genes by recombination of closely-related DNA sequences (Stemmer, 1994b). Because it also introduces new point mutations during reassembly of the DNA fragments, DNA shuffling alone has been effective for directed protein evolution starting from a single sequence Stemmer 1994a, Crameri et al 1996. Questions arise as to how this approach is best implemented and integrated with other in vitro evolution approaches such as sequential random mutagenesis. Issues include optimizing the point mutagenesis rate associated with DNA shuffling, determining appropriate screening sample sizes and how many parental genes to recombine, and deciding when to use recombination. Here we investigate the further evolution of pNB esterase by DNA shuffling of the improved sequences generated by random mutagenesis and screening. By following how the genes evolve during cycles of DNA shuffling and screening, we can elucidate the mechanisms contributing to the evolution of function and begin to optimize strategies for in vitro evolution. An analysis of the recombination process identifies some of its benefits and limitations for directed evolution and allows a rational choice of mutagenesis and screening strategies.

Section snippets

Recombination statistics and screening requirements

To comment on the utility of DNA shuffling in directed evolution, a review of the statistics of recombination of multiple parent sequences is useful. For this discussion, we will assume that the mutations are unique and distributed far enough from one another on the genes that recombination occurs freely between any two. Furthermore, equal amounts of the initial DNA sequences are recombined. Consider the random recombination of three parent sequences, each of which contains a si ngle mutation.

Conclusions

Recombination is an important tool for directing the evolution of proteins. Beneficial mutations can be recombined, while neutral and deleterious mutations are eliminated. The need to screen rather than select for many important enzyme functions, however, severely limits the ability to search for useful combinations. It is therefore imperative to analyze various recombination strategies. Mutagenic rates associated with the recombination process must be low so that beneficial mutations are not

DNA shuffling

DNA shuffling was performed as described by Stemmer (1994b) with modifications. The 2 kb DNA fragment encoding the B. subtilis pNB esterase gene was amplified using PCR (forward primer 5′-CAATCTAGAGGGTATTAATAATG-3′ and reverse primer 5′-CGCGGGATCCCCGGGTACCGGGC-3′). The amplified DNA was purified by gel electrophoresis and extraction using Qiaex kit (Qiagen, Chatsworth, CA). A total quantity of ∼10 μg DNA, either from a single parent (non-recombinatorial) or from a mixture of multiple parent

Acknowledgements

The authors thank Dr W. P. C. Stemmer (Maxygen) for many helpful discussions, Dr Steve Queener (Eli Lilly & Co.) for providing us the wild-type pNB esterase and the challenge to evolve it, and Ms Rebecca Little (Eli Lilly & Co.) for assistance with DNA sequencing. This research is supported by the US Department of Energy’s program in Biological and Chemical Technologies Research within the Office of Industrial Technologies, Energy Efficiency and Renewables. O. K. is supported by an NIH

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Edited by J. Wells

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Present addresses: J. C. Moore, Merck & Co., Inc., P.O. Box 2000, RY80Y-110, Rahway, NJ 07065, USA; H.-M. Jin, SmithKline Beecham, Mail Code UE0548, 709 Swedeland Rd., King of Prussia, PA 19406, USA.

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